Method of insulin production

a technology of insulin and production method, which is applied in the field of preparing insulin, can solve the problems of increasing the procedural complexity and the increase in the production cost, and achieve the effects of reducing the cost of removing impurities, effective control, and improving the efficiency of insulin purification

Inactive Publication Date: 2018-10-11
HANMI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]The method of the present invention can prepare an insulin sample, where impurities are effectively controlled, and thus can significantly improve insulin purification efficiency. Accordingly, the method of the present invention can be applied to large-scale production of insulin, and is thus capable of reducing cost for removing impurities.

Problems solved by technology

However, when insulin is prepared by this method, impurities, which are difficult to remove by the general purification method such as using a column, etc., in particular, a type of human insulin wherein the last amino acid in the B-chain, threonine, is deleted [Des-Thr(B30)-insulin], are mostly formed in large quantities (4% to 10%), compared with other impurities generated during insulin production, although the content of the impurities varies according to the conditions.
However, these methods may give rise to potential problems due to additives during the purification process performed following the enzyme conversion process.
Additionally, these methods may also have a problem in that a step of adding an additive to inhibit the generation of the Des-Thr(B30)-insulin and / or unblocking the additive is further required in the above process, thereby increasing the procedural complexity and leading to an increase in the production cost.

Method used

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  • Method of insulin production
  • Method of insulin production
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Examples

Experimental program
Comparison scheme
Effect test

example 1

n of Proinsulin Analogs

[0139]The expression of recombinant proinsulin analogs was performed under the regulation of T7 promoter. The sequences corresponding to insulin of each analog are shown in Table 1 below.

TABLE 1 SEQ IDAnalogSequenceNOAnalog 1DNATTC GTT AAC CAA CAC TTG TGT GGC TCA CAC CTG GTG GAA GCT5CTC TAC CTA GTG TGC GGG GAA CGA GGC TTC TTC TAC ACA CCCAAG ACC CGC CGG GAG GCA GAG GAC CTG CAG GTG GGG CAG GTGGAG CTG GGC GGG GGC CCT GGT GCA GGC AGC CTG CAG CCC TTGGCC CTG GAG GGG TCC CTG CAG AAG CGT GCG ATT GTG GAA CAATGC TGT ACC AGC ATC TGC TCC CTC TAC CAG GTG GAG AAC TACTGC AAC Protein Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala6Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr ProLys Tnr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln ValGlu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro LeuAla Leu Glu Gly Ser Leu Gln Lys Arg Ala Ile Val Glu GlnCys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn TyrCys Asn Analog 2DNATTC GTT AAC CAA CAC TTG TGT GGC TCA CAC CTG GTG G...

example 2

and Refolding of Recombinant Proinsulin Analogs

[0142]In order to change the recombinant proinsulin analogs expressed in Example 1 to a soluble form, the cells were crushed and the analogs were refolded. The cell pellets, in the amount of 170 g (wet weight), were respectively resuspended in 1 L of a solubilizing buffer solution (50 mM Tris-HCl (pH 9.0), 1 mM EDTA (pH 8.0), 0.2 M NaCl, and 0.5% Triton X-100). The cells were crushed using M-110EH (Model M1475C, AC Technology Corp.), a microfluidizer processor, under a pressure of 15,000 psi. The crushed cell lysates were centrifuged at 4° C. at 12,000 g for 30 minutes and the supernatant discarded, and the pellets were respectively resuspended in 1 L of a washing buffer solution (0.5% Triton X-100, 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, and 1 mM EDTA). The resultants were centrifuged at 4° C. at 12,000 g for 30 minutes and the pellets were respectively resuspended in distilled water, and centrifuged in the same manner. The pellets were c...

example 3

ion by Cation Exchange Chromatography

[0143]A sample where the refolding was completed was attached to an SP-FF (GE healthcare, USA) column, which was equilibrated using a 20 mM sodium citrate (pH 3.0) buffer solution containing ethanol, and then the proinsulin analog protein was eluted by a linear concentration gradient from 0% to 100% using a 20 mM sodium citrate (pH 3.0) buffer solution containing 0.5 mM potassium chloride and ethanol.

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Abstract

The present invention relates to a method of preparing insulin from proinsulin comprising converting high-concentration proinsulin into insulin by enzymatic cleavage, a method of purifying insulin, and insulin prepared therefrom.

Description

TECHNICAL FIELD[0001]The present invention relates to a method of preparing insulin from proinsulin comprising converting high-concentration proinsulin into insulin by enzymatic cleavage, a method of purifying insulin, and the insulin prepared using the same methods.BACKGROUND ART[0002]The method of preparing recombinant insulin has been continuously developed from the method of preparing semi-synthetic insulin to a two-chain method and to a method of preparing insulin from proinsulin.[0003]Among the methods of preparing insulin from proinsulin, excluding the two-chain method and the semi-synthetic method, the process of converting proinsulin into insulin using trypsin and carboxypeptidase B have been used for many years [see Kemmler, W., Clark, j., Steiner, D. F., Fed. Proc. 30 (1971) 1210; Kemmler, W., Peterson, J. D., Steiner, D. F., J. Biol. Chem., 246 (1971) 6788-6791]. However, when insulin is prepared by this method, impurities, which are difficult to remove by the general pu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/62B01D15/36B01D15/32C07K1/18C07K1/20
CPCC07K14/62B01D15/362B01D15/325C07K1/18C07K1/20B01D15/363C12Y304/17002C12Y304/21001
Inventor CHOI, SEONG HOKIM, DAE JINKIM, JIN YOUNGKIM, SANG YUNCHOI, IN YOUNGKWON, SE CHANG
Owner HANMI PHARMA
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